Biodegradation Of Brewer’s Spent Grains By Aspergillus Niger And Thermomyces Lanuginosa

China is now the world’s largest beer producing country,and brewer’s grains are the main residue in the production process,and its efficient conversion is an important economic and environmental problem that large breweries need to solve.Brewer’s grains are rich in cellulose,hemicellulose and protein.Untreated brewer’s grains will quickly spoil,which not only affects the environment,but also increases the difficulty of processing.At present,the main processing method of brewer’s grains is as animal ration feed in farms.However,this method has limited digestibility and low economic benefits.Therefore,it has become a hot research direction to find a suitable method to convert the nutrients in brewer’s grains into high value-added products.Filamentous fungi are an important class of microorganisms in the environment.Filamentous fungi that can degrade plant biomass often contain a variety of lignocellulose-related degrading enzymes.Therefore,on the basis of understanding the chemical composition and spatial structure of the spent grains,and comprehensively and systematically analyzing the lignocellulose and other polysaccharide degrading enzymes of different filamentous fungi and their preferences,the appropriate fungi were selected for efficient degradation of wheat spent grains,which can realize high value-added conversion of spent grains.In this paper,the fermentative degradation and transformation of two filamentous fungi under four pretreatment methods were studied,and the dynamic changes of fungal extracellular proteins and reducing sugars during fermentation were systematically studied through genomic and proteomic systems.and achieved good phased research results on the basic research on the high-value transformation of brewer’s grains.1.The potential of Aspergillus niger An76 and Thermomyces lanuginosa SSBP to degrade brewer’s grains was analyzed by comparative genomics.Brewer’s grains are mainly composed of three parts,namely,the husk,aleurone layer and endosperm cell wall residues.The wheat husk is mainly composed of lignin and cellulose,and the aleurone layer and endosperm cell wall residues are mainly arabinoxylan and arabinoxylan.It is composed of hemicellulose such as beta-1,3-1,4-glucan and a small amount of cellulose.Through analysis,the genome of Aspergillus niger(An76)was found to encode a total of 151 lignocellulose-degrading enzymes,which not only contain complete cellulase systems,but also have various backbone enzymes for degrading hemicellulose and side chain enzymes and has a variety of redox proteins involved in lignin degradation.Therefore,Aspergillus niger An76 has the potential for efficient degradation and transformation of spent grains.Thermomyces lanuginosus(SSBP),as a dominant fungus in high-temperature aerobic lignocellulose composting,encodes 46 lignocellulose degradation-related genes in its genome,including 7 cellulose degradation-related β-Glucosidase,exo-cellulase and endo-cellulase genes;encoding 19 β-1,3-1,4-glucan-related degrading enzymes and 13 galactomannan-related degrading enzymes Tie.;For the degradation of xylan,the bacteria only encodes one endo-xylanase and one xylosidase,but no exo-and endo-cellulase genes.The genome analysis showed that for lignocellulose,Thermomyces lanuginosa could only utilize xylan and β-1,3-1,4-glucan,but could not degrade cellulose and lignin.Since the xylan-related degrading enzymes do not contain xylan side-chain degrading enzymes,there may be accumulation of xylo-oligosaccharides with side-chains during the fermentation of brewer’s grains with Thermomyces lanuginosa.2.Using the method of functional omics,the differences of the extracellular degrading enzymes of Aspergillus niger after different pretreatments were analyzed,and it was found that the growth of Aspergillus niger was the best on the straws pretreated by steam explosion.The brewer’s spent grains were subjected to pretreatment such as mechanical grinding,alkali treatment and steam explosion.The pretreated and untreated four kinds of spent grains were selected for the fermentation of Aspergillus niger.Grows best.Ultrastructural analysis showed that steam explosion treatment destroyed the supramolecular cross-linked structure of brewer’s spent grains,resulting in partial destruction of lignin,which improved the accessibility of cells to cellulose and hemicellulose components and the specific surface area of contact,so that the fermentation process of Aspergillus niger was significantly advanced.In the culture of steam-exploded wheat spent grains as substrate,39 lignocellulose-related degrading enzymes were detected in the secretome of Aspergillus niger,including 10 xylanases,5 β-1,3-1,4-Glucanase and 13 cellulose degrading enzymes,a total of 18 glycoside hydrolases with relative secretion higher than 1%.Using fluorescence-assisted sugar electrophoresis analysis,it was found that a large number of monosaccharides and oligosaccharides were produced in the fermentation base after 3 days of fermentation,and these monosaccharides and oligosaccharides were rapidly utilized by the bacteria in the later stage.Using the crude enzyme liquid of Aspergillus niger obtained by fermentation and extraction of malted grains,the polysaccharide components in malted grains can be degraded to obtain oligosaccharides and monosaccharides.3.Using the method of functional omics,the expression differences of the extracellular enzymes of Thermomyces lanuginosa cultured on different pretreated grains were analyzed.The strain also grew best on the pretreated grains by steam explosion.The detection and analysis of the fermentation process of Thermomyces lanuginosa on four different pretreated brewer’s spent grains showed that Thermomyces lanuginosa also grew best on the steam-exploded spent grains.Twenty-eight lignocellulose-related degrading enzymes were detected in its secretome,except for four enzyme components,including one xylan backbone degrading enzyme and one xylosidase,whose secretion ratio was higher than 1%,there is a GH17 family of β-1,3-glucanase and another GH3 family of cellulose-degrading glucosidase relative secretion is relatively high.In terms of the ability of the bacteria to degrade the fermentation process of wheat spent grains,the enzymes secreted by the bacteria have the activity of degrading xylanase and β-1,3-1,4-glucan.Under the action of β-1,3-1,4-glucanase,xylan and β-1,3-1,4-glucan in spent grains can be degraded into oligosaccharides.Fluorescence-assisted sugar electrophoresis analysis found that a large amount of xylo-oligosaccharides and β-1,3-1,4-glucose-oligosaccharide oligosaccharides existed in the late stage of fermentation,indicating that the use of Thermomyces lanuginosa was not complete due to the incomplete enzyme system.Relevant oligosaccharides are degraded into monosaccharides and utilized.The crude enzyme liquid of Thermomyces lanuginosa obtained by fermentation and extraction of brewer’s spent grains can degrade the hemicellulose polysaccharide components in the brewer’s spent grains to obtain oligosaccharides when the brewer’s spent grains are used as the substrate.4.The xylanase XynA and β-1,3-1,4-glucanase Glu16A in Thermomyces lanuginosa were heterologously expressed,and the two recombinant enzymes could effectively analyze their effects in the degradation of beer wheat.The ability of xylan and β-1,3-1,4-glucan in grains to produce oligosaccharides.Successful heterologous expression of the xylanase XynA and β-1,3-1,4-glucanase Glu16A from in Escherichia coli,the basic enzymology of the two recombinant enzymes The properties were characterized.The results showed that the optimum temperature of XynA was 70℃,and the optimum pH was 6.0;the optimum temperature of Glu16A was 60℃,and the optimum pH was 10.0.The enzyme stability of Glu16A at low temperature was better,and has a wide pH range.Recombinant xylanase XynA can degrade beech xylan,and the product is xylan oligosaccharide;recombinantβ-1,3-1,4-glucanase Glu16A can degrade β-1,3-1,4-glucan sugars,resulting in beta-oligosaccharides containing beta-1,3 and beta-1,4 glycosidic linkages.Oligosaccharides can be produced by degrading the spent grains using two recombinant enzymes,XynA degrades the arabinoxylan in the spent grains into a variety of oligosaccharides,including xylo-oligosaccharides with side chains and xylo-oligosaccharides without side chains,Glu16A degrades grain β-1,3-1,4-glucan in brewer’s grains into oligoglucosaccharides containing β-1,3 and β-1,4 glycosidic bonds.The results have laid a theoretical foundation for the next step in the production of oligosaccharides from brewer’s spent grains.